Disulfide bond formation between polypeptides of a protein is a critical aspect of proper protein assembly and structure. Protein multimerization may be dependent on sufficient levels of disulfide bond formation between polypeptides (e.g. polypeptide monomers/multimers). Antibodies represent one class of proteins that is particularly dependent on disulfide bond formation.
Antibodies are generally composed of four polypeptides, two light chains and two heavy chains (L:H:H:L). Most antibodies contain disulfide bonds between the four polypeptide chains. Occasionally, the disulfide bonds between the heavy chain polypeptides are not formed, resulting in the formation of an antibody comprising no interchain disulfide bonds between the two pairs of heavy and light chains. See, generally, FIG. 1. These antibodies have been termed half antibodies (abbreviated herein as “Hab”).
Certain antibody classes and types are more susceptible to half antibody formation, such as the immunoglobulin G, subclass 4 (abbreviated herein as “IgG4”) antibodies. In both natural and recombinant antibody production, a significant proportion of IgG4 antibodies, at least as high as 35%, are produced as half antibodies. (Miesages et al., 2012, Biotechnol Bioeng. 109(8): 2048-58).
Under physiological conditions, half antibodies typically exist as complete, functional antibodies due to strong non-covalent interactions between the two heavy chain-light chain antibody halves, despite the absence of interchain disulfide bonds. (Rose et al., 2011, Structure, 19(9):1274-82). Half antibody formation has been associated with formation of aberrant proteins (See FIG. 1). For example, half-antibody formation in IgG4 antibodies may be due to the primary amino acid sequence and structure of the hinge region, which results in the ability of IgG4 antibodies to perform dynamic Fab arm exchange in which two antibodies can recombine with one another to form bispecific antibodies. (See, e.g., U.S. Pat. No. 4,470,925; U.S. Patent Application Publication No. US 2011/0086366 A1). This feature of the IgG4 subtype may be accounted for by the increased flexibility of the hinge sequence that makes it easier to form the cyclic intra-chain disulfide bond. (Bloom et al., 1997, Protein Sci. 6(2):407-15; Schuurman et al., 2001, Mol Immunol. 38(1):1-8). Half antibody formation may also be traced to deletions in the heavy chain constant domains, such as with antibodies produced by certain myelomas (Spiegelberg et al., 1975, Biochemistry 14(10):2157-63).
Half antibodies are not currently known to be associated with any distinct clinical syndrome or toxicology. However, the level of half antibody is a critical quality attribute for the production and/or manufacturing of therapeutic IgG4 antibodies. The extent to which upstream or downstream antibody processing conditions can be used to control the formation of disulfide bonds between polypeptides of multimeric proteins in final drug substances is poorly understood.